Method for the separation of protein from animal matter containing protein in water-soluble form



Patented-M51945 amnion role m ssrm'rron 0F mo- THIN mom MATTER CONTAIN ING PROTEIN IN WATER-SOLUBLE FORM .Psesuirno William G. Gordon; Philadelphia to Smitlalline & French laboratories, Philadelpliia, 2a., aoorporation oi Pennsylvania 's cation September a, 194:,

s21: No. sums 13 Claims. (Cl- 260-112) 'lhisinventionrclatestoamethodiortheseparationandrocoveryoiproteiniromanimalmat- 'tercontainingproteinrecoverableinwater-solubleiorm.

'l'hemethodaccordingtothisinventionisefllo cisnt and econmnical the protein product willherccoveredin undenatured,water-soluble,

heatcoasulableiormandvariouslyadaptableior ioodandcommercialuse 'ihemethodaccordinztothisinventioninvolves 1o essentiallyprecipltationoiproteiniromanimal matter taininz oi'themotap nsutt r-means accanplished by dissociationoifi protein metaphosphate.

* 4 andrecoveryoiproteinirom'solutl'onunderconditionstoavoiddenaturationoitheprotein.

l lore specifically. preferable procedure accordins tothis invention will comprise precipitation oi as whey, bovine sermandthelikabymetaphosphoric acid, disprotein-metaphosphate, in suspensioninwatenthroughthemediumoianalkali, 3'0

with the production at protein in solution-and oi alkali metaphosphate partly precipit d and 'proteinsolutioniorrecove'ryoitheprotein.

In proceeding with the initial treatment of animalmatter,asbovineserum,andthelike,-iorthe 4o precipitation protein,anysuitablesourceoi thelikaianacidwillbc-added torelease metaphosphoric orlnorsa'nie acid 1 maybeinsdiorthispm'posesolonsasit doesnotss itseli form a water-insoluble compound with proteinandisoisumcientstrengthtoproducethe desired pH value, which will appear, tor the precipitation oi the protein as protein metaphosphate. Thus, for example, but without limitation upon the term acid, hydrochloric, sulphuric.

nitric, phosphoric, acetic acids will be eiiective. .It may be noted that where the protein product isintendedioruseasiood,itwillbedcsirableto use an acid which does not produce toxic eflects. To eiiect the precipitation 0! protein through 4 the medium of metaphosphoric acid, the pH value of the animal matter will be adjusted 'to within about the range pH 4.3-pH 1.0. or within the narrower range pH 3.5-pI-I 1.8, and preferably to about pH 3.0. The adjustment of the pH value will, of course, be accomplished through the medium or the metaphosphoric acid it such be used, ortheaeidusedinconnectionwithasource oi metaphosphoric acid, it such be used.

The precipitated protein metaphosphate after separation, as by centriiuzation,"filtration, or the like, and washing with water, is suspended in v .wateranddissociated by treatment withanalkali.

In this step any alkali, which itseli will not form a water-insoluble precipitate with protein, may be used. However, the alkali will preferably be one which will form a water-insoluble metaphosphate. Without limiting the term "alkali," for example,

calcium 'hydroxlde. barium hydroxide will be foundtobeeflectiv. Alkalissuchassodium, potassium and ammonium hydroxides, carbonates andbicarbonatesmaybeused,butsuchwillproduoe water-soluble metaphosphates, and it will be necessary to dialyze the protein solution to remove metaphosphate ion. Where the'protein is intended for food use the alkali will desirably be one which is non-toxic.

The dissociation of the protein mctaphosphate through the medium of an alkali will be accomplished with adjustment oi the pH value of the vehicle, (water) vto within about the range pH Bil-p312, or within the narrower range pH 8- pH 9.75 and preierably to about pH 9.0.

The dissociation of the protein metaphosphate will produce protein insolution and, "when an alkali which does not form a water-soluble meta phosphate is used. alkali metaphosphate' partly precipitated and partly in colloidal solution.

so When a water-soluble alkali metaphosphate is produced the metaphosphate ions are removed by aialysis, leaving the protein in pure form in soluon. When the alkali metaph sphateis partly precipitatedandpartlyincolloidalsolution, andthe use of an alkali which will produce a water-insoluble metaphosphate is greatly preferred, the alkali metaphosphate in colloidal solution is flocculated, by treatment of the mixture with an acid. The acid may be any acid of a strength sufllcient to produce the desired pH value, as will appear, and which will not itself form a water-insoluble compound with protein. By way of example, without other limitation upon the term acidf hydrochloric, nitric, acetic, lactic acids will be efiectlve. The acid will preferably be a non-toxic acid where the protein is intended for food use.

Flocculation of the alkali metaphosphate will be efiected by the addition of the acid. Desirably, though not necessarily, the acid will be used in amount to bring the pH value of the vehicle within about the range pH 1.0-pH 5.5.

The protein solution is then brought; to a pH value of about P 6.5-pH 8.0 by the addition of any alkali which will not combine with the protein with formation of a water-insoluble compound, preference being had for one which is nontoxic when the protein product is intended for food use. By way of example, but without limitation on the term alkali," sodium, potassium, ammonium and calcium hydroxide. carbonate, or bicarbonate will be effective.

After neutralization of the solutlon'the flocculated alkali metaphosphate, which settles out as a gelatinous precipitate, i 'removed, by centrifuging, fiitering or otherwise, and washed with water, the wash water being desirably retumedto the protein solution.

The protein solution is then dried in any suitable manner with avoidance of denaturation of the water-soluble protein.

As a specific illustration of procedure according to this invention, for the recovery of protein from, for example, whey, there is mixed with a quantity of acid whey, having, for example, a' pH value of about pH 4.2, sufficient sodium metaphosphate or hexametaphosphate to react with the protein content of the whey and nlfl'icient hydrochloric acid to efiect adjustment of the pH value of the mixture to about pH 3.0, with precip itation of protein metaphosphate. The order of the addition of the alkali hexametaphosphate, or alkali metaphosphate, and of the acid to the whey is not of primary importance.

However, it will be found that if the acid be first added and then the alkali hexametaphosphate, or alkali metaphosphate, settlement of the precipitate will be greatly facilitated.

The amount of sodium hexametaphosphate to be added to any given quantity of whey may be readily estimated from the fact that in general theprotein in 100 cc. of whey will be reacted with about 0.2 gram of sodium hexametaphosphate. J

The amount of acid required to adjust the pH value to about 3.0 will be readily determined on the basis of the pH value of the whey and the amount of alkali hexametaphosphate, or alkali metaphosphate, used. v y

' The precipitated protein metaphosphate will be separated from the solution in any convenient manner. as, for example, by centrifuging, filtering, or the like, and will desirably be washed with water.

To the separated protein metaphosphate will be added aquantity of water and calcium hydroxide in amount somewhat in excess of that which wouldbe chemically equivalent to the amount of metaphosphate (P0: ions) originally used, and then hydrochloric acid will be added to effect adtle out as a gelatinous precipitate. The pH value of the solution is then adjusted to apH value of about pH 6.5-pH 8.0, preferably about pH 7.0, by the addition of sodium hydroxide solution.

After adjustment of the pH value of the solution to pH'6.5-pH 8.0 the precipitated calcium metaphosphate will be removed by centrifuging, filtering, or the like, and desirably the precipitate will be washed with a small quantity of water. to

recover the adhering protein solution, which will be returned to the original solution. I

The protein solution is then dried, for example,

by heating to. a temperature of 45-50" C. under a pressure of about 20-40 mm. mercury. The dry protein obtained will be in solid form and, as will be obvious, may be ground to powder. Alternatively. the protein solution may be spray dried with initial production of the protein in powdered form, or by any other suitable method which will not cause heat denaturation of the protein.

As a further specific example of procedure according to this invention for the recovery of protein from, for example, bovine serum, to about milliliters of bovine serum containing 6.5 grams protein there is added about 2 milliequlvalents (.204 gram) of sodium hexametaphosphate per gram of protein and sumcient hydrochloric acid to efiect adjustment of the pH value of the mixture to about pH 3.0 with precipitation ofprotein metaphosphate. v

The order of addition of theaikall hexametaphosphate and of the acid to'the bovine serum is not of great importance. However, if it be found that the acid be first added, then the hexametaphosphate, settlement of the protein precipitate.

will be greatly facilitated.

The precipitate. protein metaphosphate, is separated, as, for example, by centrifuging, filtering, or the like, and will desirably be washed with water.

The precipitate will then be suspended in about 100 milliliter of water and to the suspension will be added milligrams (.150 gram) of calcium hydroxide per 2 milllequlvalents (.204 gram) of .metaphosphate originally used. The calcium hydroxide will raise the pH of the suspension to about pH 10.5. Hydrochloric acid is then immediately added to the suspension in amount sufthe solution to about pH 5.0, when the calcium m taphosphate will flocculate and settle out as a gelatinous precipitate. After the solution has stood for a few minutes the pH value is adjusted to about pH 7.0 by the addition of sodium hydroxide solution to complete the flocculation.

'dl'ledinmlilitable When in. mum is was. precipitatedcalciumphosphatewillberemovedbycen.

trlfllginmfllterinmorthelikaanddesirablythe precipitatewillbewashedwithasmallquantity water to recover adheringprotein solution, which will be returned to the original solution. The protein solution thus obtained is then dried byheatingto a temperature oisay 40-50 O.un-

.will be in undenatured, water-soluble, heat coagulable form.

It will be understood that it is not intended that this invention be limited to the details variously involved in the above description of the method and its carrying out, given for illustrative purposes, since it is contemplated that the procedure maybe variously modified in detail without departing irom the scope of the invention as defined in the appended claims.

'This application is a continuation-in-part of application flied by me, Serial No. 424,125, filed December 23, 1941, which in turn is a continuation-in-part of application filed by me, Serial No. 378,944, filed February 14, 1941.

What I claim and desire to protect by Letters Patent is:

1. In a method of separating protein from aniinal matter containing protein recoverable in waprecipitate in the presence of water containing .an alkali which will form a water-insoluble compound with metaphosphate ions and recovering protein from the water.

2. In a method oi separating protein irom sociating the precipitate with an aqueous alkali solution; which will produce a water-insoluble compound with metsphosphate ions, fiocculating metaphosphate formed, separating fioccul ated alkali metaphosphate iron: the water and recovering soluble protein from the water.

8. In a method of separating protein from whey-the steps'comprising precipitating protein irom whey by means of metaphosphoric acid, separating metaphosphate radical from-the precipitate by means oi an aqueous solution of an alkali which will form a water-insoluble compound with metaphosphate ions, adding to the solution an acid which will not form with protein a water-insoluble compound to efiect fiocculation oi alkali metaphosphate formed, separating fiocculated alkali metaphosphate and recovering'soluble protein from the water. a '7. In a method of separating protein 1 whey the steps comprising precipitating protein from whey by means of metaphosphoric acid, separating metaphosphate radical from the precipimetaphosphate and tateinthepresenceoiwaterbymeansoianalkali which will form. a water-insoluble compound with metaphosphate ions, fiocculating alkali metaphosphate formed by means of an acid which will not react with protein to form a water-insoluble compound in amount-such as to adjust the pH value of the water to within about the range pH 5.5-pI-I 1.0, separating fiooculated alkali recovering soluble protein from the water.

8. In a method of separating protein irom whey the steps comprising precipitating protein from whey having a pH value within about the range pH 4.3-pH 1.0 by means of metaphosphoric acid, separating metaphosl hote radical from the precipitate in the presence of water by means of an alkali which will form a water-insoluble compound with metaphosphate ions, under conditions of hydrogen ion concentration within about the range pH 6.0 to pH 12.0, fiocculating alkali metawhey the steps comprising precipitating protein pH 4.3-pH 1.0 by means oi metaphosphoric acid,

dissociating the precipitate in the presence of water containing an alkali which will form a ater-insoluble compound with metaphosphate 1 ns and recovering protein from the water.

4. In a method of separating protein from whey the steps comprising precipitating protein from whey by means of metaphosphoric acid, dissociating the precipitate by means of an alkali solution which will produce a water-insoluble compound with metaphosphate ions under conditions oi hydrogen ion concentration within about the range pI-I 6.0-pH 12.0, separating alkali metaphosphate formed and recovering soluble protein from the water.

5. In a method 0! separating protein from whey the steps comprising precipitating protein irom whey by means oi metaphosphoric acid, dishosphate formed by means oi an acid which will' ot react with protein to form a water-insoluble compound in amount such as to adjust the pH value of the water within about the range pH 5.5-pH 1.0, separating fiocculated alkali metaphosphate and recovering soluble protein irom the water.

9. In a method of separating protein from whey the steps comprising precipitating protein from whey at a pH value within about the range pH 4.3-pH 1.0 by means of metaphosphoric acid. separating meta-phosphate radical from the precipitate through the medium of calcium hydroxide in the presence of water, under conditions of hydrogen ion concentration within about the range pH 6.0-pH 12.0, fiocculating the calcium metaphosphate formed by means of hydrochloric acid in amount to'adlust the pH value 01 the water to within about the range pH 5.5 to pH 1.0, neutralizing the solution, separating flocculated calcium metaphosphate and drying the solution of protein in water. 10. In a method of separating protein iro bovine serum the steps comprising precipitating protein from bovine serum by means of metaphosphoric acid, dissociating the precipitate in the presence or water containing an alkali which will form a water-insoluble compound with metaphotzphate ions and recovering protein from the we r.

, 11. In a method of separating protein from bovine serum comprising precipitating protein from bovine serum by means of metaphosphorie soluble protein from the water.

12. In a method of separating protein from bovine serum the stepscomprising precipitating. i

protein from bovine serum having a .pH value within about the range pH 4.3-pH 1.0 by means of .metaphosphoric acid, separating metaphosphate radical from the precipitate in the presence of water by means of an alkali which will form a water-insoluble compound with metaphosphate ions, under'conditions of hydrogen ion concentration within about the range pH 6.0-pH 12.0,

flocculating alkali metaphosphate formed 'bymeans of an acid which will not react with protein .to' form a water-insoluble compound in amount such as to adjust the pH value of the water within about the range pH 5.5-pH 1.0, separating flocculated alkali metaphosphate and recovering soluble protein from the water.

13. In a method of separating protein from bovine serum the steps comprising precipitating protein from bovine serum at a pH value within about the range pH 4.3-pH1.0 by means of metai0 phosphoric acid, separating metaphosphate radical from the precipitate through the medium of calcium hydroxide in the presence of water, under conditions of hydrogen ion concentration within'about the range pH 6.0-pH 12.0, flocculatzo-the solution of protein in water.

is ing'the calcium metaphosphate formed by means oiihydrochloric acid in amount to adjust the pH value oithe water to within about the range pH 5.5-pH 1.0, neutralizing the solution, separating flooculated' calcium metaphosphate and drying WILLIAM G. GORDON.- 

